Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Comparison of rhizobitoxine-induced inhibition of β-cystathionase from different bradyrhizobia and soybean genotypes

The enzyme -cystathionase catalyzes the conversion of cystathionine to homocysteine in both plants and bacteria. Preparations of this enzyme taken from both Salmonella and spinach (Spinacia oleracea L.) have been shown to be irreversibly inhibited by low concentrations of rhizobitoxine (RT), a chlorosis-inducing phytotoxin produced by some strains of soybean bradyrhizobia. The sensitivities of -cystathionase from bradyrhizobia and soybean are not well characterized. Therefore, we purified -cystathionase from selected bradyrhizobia and soybean genotypes that have been shown to exhibit differences in RT production and apparent RT sensitivities, respectively. Enzyme purified from E. coli strain DH52 was used for comparison. The enzymes differed in their physiological properties and RT sensitivities. Overall, the -cystathionase enzymes purified from bradyrhizobia were more sensitive to RT than were those from the soybean cultivars. Kinetic studies showed that the nature of the RT-induced inhibition also differed between the two sources. The enzymes from bradyrhizobia exhibited inhibition that was [RT]-dependent, whereas the enzymes from soybean showed a time-dependent inhibition. These contrasting characteristics may in part reflect differences in active site accessibility, amino acid components, and associated RT diffusion rates. However, in all cases the inhibition caused by RT showed a typical substrate-competitive inhibition pattern.Abbreviations RT rhizobitoxine - PLP pyridoxal phosphate - DTT dithioerythritol - PMSF phenylmethylsulfonyl fluoride - HTP hydroxyapatite Published as Paper No. 1571 in the Journal Series of the Delaware Agricultural Experiment Station.     

Effects of Lovastatin and Pravastatin on Amyloid Processing and Inflammatory Response in TgCRND8 Brain

Previous studies suggest that treatment with statins reduce beta amyloid (Abeta) deposition in brains of mouse models of Alzheimer's disease (AD) and may reduce the prevalence of AD in humans. Since lipophilicity influences the biological efficacy of statins, we compared the effects of lovastatin, a lipophilic statin, to effects of the hydrophilic pravastatin on amyloid processing and inflammatory responses in brain. Three-month old TgCRND8 mice expressing mutant human amyloid precursor protein (mHuAPP) were treated daily with various doses of either statin. After 1 month, levels of cerebral soluble and fibrillar Abeta peptides, soluble sAPPalpha, and inflammatory cytokines were measured. Both statins caused dose-dependent reductions in total Abeta peptides with parallel increases in total sAPPalpha. At all doses, slightly greater effects were observed with lovastatin than with pravastatin. In contrast, only lovastatin significantly increased levels of IL-1beta and of TNFalpha in a dose-dependent manner. Lovastatin, but not pravastatin, decreased succinic dehydrogenase and increased lactate dehydrogenase activities in skeletal muscle and increased TUNEL staining in liver. Our data demonstrate that both statins shift the balance of APP processing from excessive beta-toward the normal alpha-cleavage while reducing the total amyloid burden in TgCRND8 brain and that lovastatin, but not pravastatin, potentiates cerebral inflammation and is associated with liver and muscle histotoxicity in these animals. These data show that pravastatin can reduce amyloid burden without potentiating inflammatory responses in brain and, therefore, may have a wider dose-range of safety than have lipophilic statins in the treatment or prevention of AD.     

Formylation of porphyrin platinum complexes

The formylation reaction of platinum complexes of -unsubstituted porphyrins was studied. The interaction of deuteroporphyrin IX derivatives with the Vilsmeyer reagent led to the selective formylation of their macrocycles in the position. The resulting formyl derivatives of the porphyrins are of interest for fluorescent immunoassay.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 103–107.Original Russian Text Copyright © 2005 by Rumyantseva, Konovalenko, Nagaeva, Mironov.     

Chemical modification of Interleukin-1β: Biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein

We have modified recombinant human Interleukin-1 using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide atpH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI=6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys₈₈ and Lys₉₂). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu₁₁₁ and Lys₁₃₈ were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 and may be important for the binding of IL-1 to its receptor.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside